The fate of any lineage is contingent on the rate at which its genome changes over time. Genome dynamics are influenced by patterns of mutation and recombination. Mutations as the raw force of variation can be acted on independently during exchanges of homologous genetic regions via meiotic recombination. While molecular evolution in sexual lineages is impacted by both mutation and recombination, asexual lineage fate is primarily influenced by the mutation rate; recombination is often altered or absent in asexuals. Although multiple studies show accelerated mutation accumulation in asexual lineages that have lost recombination, virtually nothing is known about rate patterns when meiosis is retained. Here, I use parasitic wasps in genus Diachasma to investigate genome evolution in a recently-derived asexual lineage. I provide evidence that asexual Diachasma possess a canonical set of meiosis genes as well as high levels of genomic homozygosity. Taken together, these observations support an active, albeit modified, form of meiosis in this asexual lineage. In addition, I present the first documentation of accelerated mutation accumulation in the nuclear genome of a naturally-occurring, meiotically- reproducing organism. If harmful, these mutations could impede asexual lineage persistence and contribute strong support for the long-term benefits of sex.

Orientador: José Paes de Oliveira Filho / Resumo: O Labrador Retriever (LR) é uma das principais raças caninas com maior predisposição à obesidade. Vários fatores, especialmente as inter-relações entre a alimentação, exercício e fatores sociais, influenciam a probabilidade de um cão se tornar obeso. Além disso, fatores genéticos são também responsáveis pela obesidade em cães, e no LR a mutação P187fs no gene pró-opiomelanocortina (POMC) está fortemente associada à obesidade. Não existem estudos prévios de prevalência da desta em LR no Brasil. Portanto, o objetivo deste estudo foi investigar esta mutação em cães da raça LR no Brasil. Dos 108 LR avaliados neste estudo, 59 eram de um estudo retrospectivo (composto por animais atendido no hospital veterinário e sem linhagem conhecida) e 49 eram de um estudo prospectivo (composto por 19 cães pet e 30 cães de assistência/resgate). Um questionário de risco de obesidade modificado foi aplicado nos tutores dos animais usados no estudo prospectivo. Fragmentos de DNA, contendo a região mutada, foram amplificados por PCR e submetidos ao sequenciamento gênico direto. A frequência alélica da mutação (21,3%) estava fora do equilíbrio de Hardy-Weinberg (P < 0,05). Usando somente os dados dos animais de linhagem conhecida, o alelo mutado foi mais frequente no grupo de assistência/resgate que no grupo de pets (P < 0,01). Embora a mutação tenha aumentado a motivação pelo alimento em LR do grupo de assistência/resgate, outras variáveis, especialmente o frequente exercício, favoreceu a manutençã... (Resumo completo, clicar acesso eletrônico abaixo) / Abstract: The Labrador retriever (LR) is among the main breeds with the greatest predisposition to obesity. Several factors; especially the interrelationships between food management, exercise and social factors; influence the likelihood of a dog becoming obese. Furthermore, genetic factors are also responsible for obesity in dogs, and in LR, the mutation P187fs in pro-opiomelanocortin (POMC) gene is strongly associated with obesity. There is no knowledge of studies that have previously evaluated the prevalence of this mutation in Brazilian LR. Therefore, the objective of this study was to investigate this mutation in LR in Brazil. Of the 108 LR that were assessed in this study, 59 were from a previous study, composed by animals assisted in a veterinary hospital with unknown lineage, and 49 were from a prospective study, composed of 19 pet and 30 assistance/rescue LR dogs. The obesity risk and appetite questionnaire were applied, with some modifications, to tutors of the animals used in the prospective study. Fragments of the DNA, containing the mutated region, were amplified by PCR and submitted to direct gene sequencing. The mutated allele frequency (21.3%) was out of Hardy-Weinberg Equilibrium (HWE) (P < 0.05). Using only the data of animals with known lineage, the presence of the mutated allele was higher in the assistance/rescue dogs than pets (P < 0.01). Although the mutation has increased the food-motivation in the assistance/rescue dogs, other variables, especially frequent exe... (Complete abstract click electronic access below) / Mestre

To maintain genomic integrity, all cells must accurately duplicate their genetic material in order to provide intact and complete copies to each daughter cell following cell division. Successful inheritance of chromosomal information without changing even a single nucleotide requires accurate and robust DNA replication. This requires that cells tightly control replication initiation from the origin(s), processive elongation of the replisome, and the completion of DNA replication by resolving convergent replication forks ensuring that each sequence is duplicated without alteration. Unlike initiation and elongation, the process by which replication forks converge and are resolved into two discrete, inheritable DNA molecules is not well understood. This process must be remarkably efficient, occurring thousands of times per cell division in human cells, and is likely to be a fundamental step in regulating genome stability in all cells. In this dissertation I address how DNA replication completes in the model system Escherichia coli. To achieve this, I examined candidate mutants for impairments in the completion of DNA replication. By evaluating growth, viability, chromosomal copy number, and plasmid stability I identified a requirement for the proteins RecBCD, ExoI, and SbcCD in the completion reaction. SbcCD and ExoI act before RecBCD in the completion reaction and process the DNA intermediates arising as replication forks converge. These enzymes act in the completion reaction without recombination or RecA, but in the absence of the normal process recombination is required to complete DNA replication via an aberrant pathway that results in genomic instability.

Maintaining mitochondrial genome sequence integrity is essential for preserving normal mitochondrial function. Several human diseases have been associated with heteroplasmic mitochondrial genome mutations, but few genetic systems can simultaneously represent pathogenic mitochondrial genome evolution and inheritance. The nematode Caenorhabditis briggsae is one such model. Natural C. briggsae isolates are globally-distributed and phylogenetically grouped into three distinct clades, with isolates exhibiting varying levels of a large-scale mtDNA deletion, nad5∆. Furthermore, a small subset of clade II isolates exhibits putative compensatory mutations that may reduce the risk of deletion formation and accumulation in those populations. In this thesis, the author characterizes the dynamics of nad5∆ heteroplasmy levels during both development and transmission in several C. briggsae natural isolates, including two containing putatively protective compensatory mutations (C+). For all isolates tested, nad5∆ heteroplasmy levels increased across nematode development, with L1 (first larval stage) exhibiting the lowest deletion load for all but one isolate that exhibited highly variable nad5∆ levels, while the increase was slowest and overall nad5∆ levels remained relatively low in C+ isolates. These results support previous work suggesting that nad5∆ is a selfish element and demonstrate the protective nature of compensatory mutations in inhibiting mtDNA deletion accumulation. In nad5∆ inheritance assays, C+ isolates displayed a strong pattern of reversion to wildtype mtDNA levels that was not seen in isolates lacking compensatory mutations (C-). These assays also showed that nad5∆ inheritance was not well predicted by total maternal nad5∆ proportion in either C+ or C- isolates; offspring nad5∆ levels were generally much lower than maternal levels, consistent with some form of negative selection operating between generations. Assays of both maternal somatic and gonadal tissues had slightly more power to predict offspring deletion levels than did assays of whole-worm maternal samples; this result likely points to variance in deletion levels originating from an untested parental tissue type present within the whole-worm samples. This thesis provided deeper insights into the patterns of mtDNA deletion transmission and age-associated dynamics. It was the first project of its type to survey mutation dynamics and heteroplasmy levels of a naturally-occurring large-scale mtDNA deletion. Thus, this work serves to further develop C. briggsae for use as an experimental model of human mtDNA deletion dynamics and mitochondrial dysfunction.

Die Sequenzierungstechnologien entwickeln sich stetig weiter, dies ermöglicht eine zuvor nicht erreichte Ausbeute an experimentellen Daten und auch an Neuentwicklungen von zuvor nicht realisierbaren Experimenten. Zugleich werden spezifische Datenbanken, Algorithmen und Softwareprogramme entwickelt, um die neu entstandenen Daten zu analysieren. Während der Untersuchung bioinformatischer Methoden für die Identifizierung und Klassifizierung somatischer Mutationen in hämatologischen Erkrankungen, zeigte sich eine hohe Vielfalt an alternativen Softwaretools die für die jeweiligen Analyseschritte genutzt werden können. Derzeit existiert noch kein Standard zur effizienten Analyse von Mutationen aus Next-Generation-Sequencing (NGS)-Daten. Die unterschiedlichen Methoden und Pipelines generieren Kandidaten, die zum größten Anteil in allen Ansätzen identifiziert werden können, jedoch werden Software spezifische Kandidaten nicht einheitlich detektiert. Um eine einheitliche und effiziente Analyse von NGS-Daten durchzuführen war im Rahmen dieser Arbeit die Entwicklung einer benutzerfreundlichen und einheitlichen Pipeline vorgesehen. Hierfür wurden zunächst die essentiellen Analysen wie die Identifizierung der Basen, die Alignierung und die Identifizierung der Mutationen untersucht. Des Weiteren wurden unter Berücksichtigung von Effizienz und Performance diverse verfügbare Softwaretools getestet, ausgewertet und sowohl mögliche Verbesserungen als auch Erleichterungen der bisherigen Analysen vorgestellt und diskutiert. Durch Mitwirken in Konsortien wie der klinischen Forschergruppe 216 (KFO 216) und International Cancer Genome Consortium (ICGC) oder auch bei Haus-internen Projekten wurden Datensätze zu den Entitäten Multiples Myelom (MM), Burkitt Lymphom (BL) und Follikuläres Lymphom (FL) erstellt und analysiert. Die Selektion geeigneter Softwaretools und die Generierung der Pipeline basieren auf komparativen Analysen dieser Daten, sowie auf geteilte Ergebnisse und Erfahrungen in der Literatur und auch in Foren. Durch die gezielte Entwicklung von Skripten konnten biologische und klinische Fragestellungen bearbeitet werden. Hierzu zählten eine einheitliche Annotation der Gennamen, sowie die Erstellung von Genmutations-Heatmaps mit nicht Variant-Calling-File (VCF)-Syntax konformen Dateien. Des Weiteren konnten nicht abgedeckte Regionen des Genoms in den NGS-Daten identifiziert und analysiert werden. Neue Projekte zur detaillierten Untersuchung der Verteilung von wiederkehrender Mutationen und Funktionsassays zu einzelnen Mutationskandidaten konnten basierend auf den Ergebnissen initiiert werden. Durch eigens erstellte Python-Skripte konnte somit die Funktionalität der Pipeline erweitert werden und zu wichtigen Erkenntnissen bei der biologischen Interpretation der Sequenzierungsdaten führen, wie beispielsweise zu der Detektion von drei neuen molekularen Subgruppen im MM. Die Erweiterungen, der in dieser Arbeit entwickelten Pipeline verbesserte somit die Effizienz der Analyse und die Vergleichbarkeit unserer Daten. Des Weiteren konnte durch die Erstellung eines eigenen Skripts die Analyse von unbeachteten Regionen in den NGS-Daten erfolgen. / The sequencing technologies, while still being under further development, render it possible to develop novel experiments and allow the generation of larger amounts of utilizable data. At the same time novel software tools, databases and algorithms are developed to analyze these larger amounts of data. The analysis of somatic mutations in hematological malignancies showed that a high variety of alternative software tools can be used for different analysis steps. Furthermore there is currently no standardized procedure for the efficient identification and analysis of mutations in NGS data. The different pipeline and methods are, for the most part, able to identify the same mutation candidates, however there are software specific candidates which are not called by all pipelines. The scope of this dissertation was therefore to develop a user-friendly pipeline which is able to call candidate mutations uniformly and efficiently. For this purpose necessary analysis steps including base calling, alignment generation and variant calling were investigated. Furthermore available software tools were tested and evaluated regarding their efficiency and performance. Possible improvements of these software tools and previously performed analysis are explained and discussed in this work. NGS data sets of the different cancer entities multiple myeloma (MM), Burkitt lymphoma (BL) and follicular lymphoma (FL) were generated and analyzed within the framework of cooperate projects like the International Cancer Genome Consortium (ICGC) and the Clinical Research Group 216 (KFO) as well as for internal projects. The development of the pipeline and selection of suitable software tools is based on the comparative analysis of the generated data sets, as well as previously described results and experiences in literature and forums. The selective development of certain python scripts enabled the evaluation of novel biological and clinical questions by standardizing gene names in the annotation step, generating heat- maps of non-standardized VCF-files as well as the identification and analysis of uncovered regions in NGS data sets. This work and the obtained results thereby provide the groundwork for further projects e.g. the analysis of the distribution of recurrent mutations or the functional analysis of specific mutation candidates. This extensions of the developed pipeline with python scripts helped to improve the efficiency and comparability of the NGS data. The interpretation of the NGS data with the extended script for example led to the discovery of three distinct molecular subgroups in MM. Furthermore the generation of the novel python scripts helped to analyze uncovered regions in the NGS data sets.

Selection of HIV-1 variants resistant to antiretroviral therapy is well documented. However, the selection in vivo of HIV-1 mutant species that can escape host immune system HLA class I restricted cytotoxic T-lymphocyte responses has, to date, only been documented in a few individuals and its clinical importance is not well understood. This thesis analyses the observed diversity of the HIV-1 reverse transcriptase protein in a well characterised, stable, HLA-diverse cohort of HIV-1 infected patients with over two thousand patient-years of observation. The results show that HIV-1 polymorphism is selected within functional constraints and is associated with specific HLA class I alleles. Furthermore, these associations significantly cluster along the sequence and tend to occur within known corresponding HLA-restricted epitopes. Absence of polymorphism is also HLA-specific and more often seen with common HLA alleles. Knowledge of HLA specific viral polymorphisms can be used to model an individual’s viral load from their HLA type and viral sequence. These results suggest that cytotoxic T-lymphocyte escape mutation in HIV-1 is critical to the host at an individual and population level as well as to short and long term viral evolution. This work provides new insights into viral-host interactions and has clinical implications for individualisation of HIV-1 therapy and vaccine design.

No description available.

<p>Leber´s hereditary optic neuropathy (LHON), a disease affecting vision, is caused by several point mutations in mitochondrial DNA. Mutations leading to a defect NADH ubiquinone oxidoreductase protein will affect the respiratory chain and cause a disturbed ATP production. It is still unknown why this defect leads to the degeneration of retinal ganglion cells and cells in the opticus nerve as well as demyelination of axons in these areas. Analysis of mitochondrial DNA is an important tool in the diagnosis of the disease. At the present time analysis is based on cleavage by restriction enzymes, which only detects two of the most frequent mutations: m.3460G>A and m.11778G>A. This is far too few considering that more than 30 mutations are known to be associated with LHON. Therefore a new analysis method is requested. Here we describe a method based on the sequencing of the mitochondrial genes MT-ND1, MT-ND4 and MT-ND6, which will detect more than 15 different point mutations associated with the disease. To validate the analysis, DNA from 31 patients with LHON symptoms were sequenced; of these 10 were found to be positive for a LHON mutation. This result indicates that the sequencing analysis will be more effective in diagnosis of LHON than restriction enzymes.</p> / <p>Lebers hereditära optikus neuropati (LHON) är en sjukdom som beror på genetiska förändringar i arvsmassan som leder till att cellens energiomsättning rubbas. Detta gör att nervceller i ögat och synnerven bryts ned vilket leder till en synnedsättning. En patient som drabbas av LHON har inga symptom fram till dess att synen börjar försämras. Sjukdomsförloppet går snabbt och på bara några veckor är patienten ofta helt blind. Diagnostik av LHON idag utgörs av flera undersökningar av öga och synfält. Diagnosen bekräftas av en analys av arvsmassan som finns i mitokondrien, cellens energifabrik. Här beskriver vi en ny förbättrad analysmetod baserad på DNA sekvensering, dvs. bestämning av baserna i mitokondriella arvsmassan. För att utvärdera analysen har vi undersökt 31 patienter med misstänkt LHON - av dessa visade sig 10 bära på en sjuklig förändring. Resultatet visar att sekvensering med fördel kan ersätta den tidigare analysmetoden då fler sjukliga förändringar kan påvisas och utförandet av analysen är mer användarvänligt.</p>

<p>About 30 % of all infertile couples suffer from infertility of an unexplained cause. Leukemia inhibitory factor (LIF) is a glycoprotein produced by the endometrium and is an important cytokine in the implantation process. LIF exerts its biological functions through heterodimerization of its two receptors: LIF receptor (LIFR) and gp130. Point mutations in the LIF gene have been associated with female infertility. The aim of this study was to investigate whether single nucleotide polymorphisms (SNPs) in the genes of LIFR and gp130 could cause reduced fertility in women. To this end, 115 samples from women diagnosed with unexplained infertility and 191 samples from fertile women were studied. Three SNPs in the gp130 gene and two SNPs in the LIFR gene were analyzed using real-time PCR. One significant difference and a tendency to difference were detected in the gp130 gene for women with unexplained infertility. There were no differences in the LIFR gene variations. In conclusion, polymorphisms in gp130, and thereby disturbances in the LIF pathway, could be one cause for infertility in women diagnosed with unexplained infertility.</p>

In its current form, familial hypercholesterolemia (FH) is due to the presence at the heterozygous state of a mutant allele of the LDL receptor gene or of the APO gene. The patients with heterozygous familial hypercholesterolemia (HeFH) can only remove their LDL particles from the blood at about half the normal rate, leading to an approximate doubling of LDL-cholesterol (LDL-C) level, starting at birth and accumulating in many tissue resulting to tendon xanthomas, corneal arcus and early atherosclerosis (coronary heart diseases (CHD) occur typically at 35-55 years among men and at 55-75 years among women). In the early's 1990's, our interest in the field was stimulated by the fact that despite the impressive progress in the understanding of the pathophysiology and treatment of FH and a preferential r! egimen of reimbursement for statins in Belgium, many affected individuals were not diagnosed and adequately treated. In 1995, we initiated a project aimed to obtain objective information on the situation of subjects carrying familial hypercholesterolemia (FH) in Belgium and to develop better means for identifying these subjects amongst the population. During the period 1995-2006, 1305 suspected FH patients originating from 682 families were examined for a DNA-based diagnosis in our laboratory and amongst these, 544 patients (271 families) were confirmed to have FH. Seventy three mutations in the LDLR as well as the unique mutation in APOB (R3500Q) explained their FH, but amongst them, five mutations explained about 38% of FH in Wallonia and 4 mutations explained 29% of FH in Flanders. Such establishment of the spectrum of FH causing mutations and particularly, of the specific mutational spectrum by region can greatly contribute to facilitate the diagnosis of FH in the near future. In our region, we found that FH frequency is likely to be higher than the theoretical prevalence reported in most European countries (1/500). That means that, in Belgium, each GP should know at least 1 FH patient. There are several reasons that advocated for the need to precisely diagnose FH. One of which was clearly demonstrated by two of our studies: in the category of patients with very elevated cholesterol levels (VHC) and family history of early CVD (FHEC), FH patients have always more severe atherosclerosis than age-, sex-, cholesterol- and family history-matched non-FH-individuals, suggesting that the FH status is by itself a strong and independent risk factor. In this category of VHC and FHEC patients, we even found a “paradox of FH” in the fact that they have greater atherosclerosis despite relatively lower risk as predicted by the Framingham equation. Genetic test may also enhance the awareness of a greater risk than expected on the basis of other risk factors. One of the main interests of genetic tests is of course to facilitate the screening in FH family. We also developed some specific criteria in term of cholesterol levels, family history of early CHD and tendon thickening to facilitate the diagnosis of FH in Belgium. These criteria could be used to select patients susceptible to be confirmed by more expensive genetic tests. As shown by our experience in our lipid clinics, the current available lipid-lowering drugs can correct LDL-C to target in about 49% of the very high risk class of FH patients (including more than 80% FH patients of our co! hort), a real success compared to the early 1990's. In 2001, our work has contributed to the change of the reimbursement rules of statins in patients suspected of FH. Even if these new rules are not yet perfect, its positive point was that many more FH patients can now benefit of the special reimbursement compared to the situation before 2003. In conclusion, this works have illustrated many aspects regarding the existence, the frequency, the diagnostic problems and the clinical management of FH. Specially, our study suggest that FH remains underdiagnosed in despite of the fact that it is severe, can be diagnosed and effectively treated. For this reason, this work is not to be considered as a final point but rather as a “well fixed starting block” ready to support the beginning of more actions to improve the management of these FH patients at the higher level of public health.